Use of PCR analysis for airborne nucleic acids

ABSTRACT

The present invention relates to methods of analyzing airborne nucleic acid molecules using a device for the filtering and/or collecting of said molecules using an air sampling system, isolating the nucleic acids, and subsequent analysis thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of German Patent Application DE 102014 203 855.3, filed on Mar. 3, 2014.

TECHNICAL FIELD

The present invention is in the field of bioanalytics. Morespecifically, the invention relates to the detection of airborne nucleicacid molecules.

BACKGROUND

The analysis of airborne nucleic acid molecules using conventional orreal-time PCR methodologies is not currently applied worldwide due to alack of sampling techniques and data extraction methods. However, thereappear to be a variety of applications where such analytical approacheswould be useful.

In all laboratories where the use of PCR technology is routinely used, acontamination problem commonly arises during the amplification of thesame template DNA after a period of time. Due to aerosol generation(pipetting), the regular multiplication of the same nucleic acid regionsresults in amplicon contamination of the surrounding ambient air andwall surfaces. As a result, due to certain enrichment concentration overtime, false-positive results occur since the airborne nucleic acids areable to enter those samples present in new analytical vessels. Thissituation has led to a required spatial separation of master mixpreparations and the actual PCR being carried out, or to the necessaryapplication of extra technical measures to prevent contamination of thepreparations (i.e. PCR cabins). Such accumulated “contaminating” nucleicacid is to be expected not only in forensic laboratories, but also inlaboratories dedicated to inspecting genetically modified food or feed.In the context of preventive quantitative analytics, it thus would seemdesirable to routinely perform analysis of ambient air in order todetermine the presence of contaminating nucleic acid fragments.

In addition to safeguarding quality-assurance procedures, another highlyinteresting field for airborne analytics emerges in the context offorensic analysis at crime scenes. When present in a room, every humanbeing releases cellular material, and therefore clearly traceablegenetic material, into the ambient air. Depending on the mode of humanactivity, said genetic material is released in lower or higherconcentrations. Mere human presence leads to desquamation of thecornified stratified squamous epithelium of the skin, in addition tocellular material that is released into the ambient air due to coughingor sneezing reflexes via the mucous membranes; these secretions allcontain personal nucleic acid components. If it were possible toreliably achieve enrichment of these genetic materials by airfiltration, this would offer a unique forensic approach in addition toan effective searching strategy for detecting cellular or blood tracesthus evidencing the presence of a specific person at a crime scene.However, to achieve this purpose, it would thus be necessary to directlyfilter large volumes of air on-site in order to deposit as muchbiological material as possible on a filter as derived from recentlypresent persons. For example, air collection devices from themanufacturer Sartorius might enable a possibility of achievingsufficient biological concentrations on filter surfaces.

SUMMARY OF THE INVENTION

The present invention relates to a method for analyzing airborne nucleicacid molecules, the method comprising: filtering and/or collecting airusing an air sampling system comprising an air filter and/or acollection medium, such that the nucleic acid molecules remain on theair filter and in said collection medium; isolating the nucleic acidmolecules from the air filter and/or from the collecting medium; andanalysis of nucleic acid molecules, preferably by PCR and subsequent gelelectrophoresis.

The analysis can be carried out by PCR and subsequent gelelectrophoresis, PCR and subsequent MALDI-TOF mass spectrometry and/orby in situ PCR.

In one embodiment, the air filter is a membrane filter made ofnitrocellulose, wherein the pore size is from preferably 5 microns to 8microns. In another embodiment, the air filter is a gelatin filter,wherein the pore size is from preferably 2 microns to 5 microns. Inanother embodiment, the collection medium is a suitable buffer, in whichthe nucleic acids/cells are collected by impaction. In anotherembodiment, the collection medium is a surface on which the nucleicacids/cells are deposited electrostatically or otherwise.

In one embodiment, the filter/collection medium can be decomposed intoindividual fragments on a contact-free basis by means of laser ormechanical device situated in a sterile housing unit, whereby theindividual fragments are processed manually or automatically.

The air sampling system can be a Sartorius MD 8 system, an impactionsystem, or an electrostatic system.

In one embodiment, the inventive method is used for detectingcontaminating nucleic acid molecules in a laboratory room, a productionroom, a storage room, or a lounge or a meeting room. The method can beused to obtain a human DNA profile from airborne human cellularmaterial, or in the forensic analyses of crime scenes.

In one embodiment, the cellular material is dander or mucosal cells.

Furthermore, the invention also encompasses the use of an air samplingsystem comprising an air filter and/or a collecting medium for theanalysis of airborne nucleic acid molecules. In one embodiment, theinvention comprises the use of an air sampling system, comprising an airfilter and/or a collection medium for obtaining a human DNA profilederived from airborne human cell material.

DESCRIPTION OF THE FIGURES (HEREINAFTER THE FIGURE LISTING)

FIG. 1 shows processing schemes of four different embodiments of theinvention.

FIG. 2 shows an MD 8 air sampling system from the manufacturer Sartoriusfor filtration based on isokinetic sampling.

FIG. 3 shows an air filter for the MD 8 air sampling system from themanufacturer Sartorius with holders.

FIG. 4 schematically shows the principle of the analysis method usingMALDI-TOF MS.

FIG. 5 shows agarose gel electrophoresis results: (left to right)marker, extraction of nitrocellulose, extraction of gelatin, marker

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A “nucleic acid molecule” within the meaning of the invention, can beany nucleic acid molecule of any length. Examples of nucleic acids aredeoxyribonucleic acids (DNA), ribonucleic acids (RNA) or peptide nucleicacids (PNA). Nucleic acids may be single-stranded, double-stranded, orpartially single-stranded and partially double-stranded.

An “air sampling system” within the meaning of the present inventionrefers to any technical device that can be used to isolate non-gaseousconstituents from the air. In particular, collection/filtration systemsfor air are contemplated, which can collect and deposit/intercept theparticles from the air.

A “crime scene” within the meaning of the invention, is any place, e.g.closed rooms or outside, which was or likely was the scene of a crime orwhere a course of events is to be resolved.

The term “forensic analysis” refers to any investigation of a crimescene, in which the determination of the presence of a human or otherliving organism at a given moment in the past plays a role in thecriminal event.

Methods

The present invention is based on the screening of variousfilter/collection-media/materials for air sampling and subsequentextraction/processing of the filter/collection media for subsequent PCR.

One inventive method comprises the filtration/collection of air that issuspected of containing air-borne nucleic acid molecules using an airsampling system that comprises: an air filter/a collection medium suchthat the nucleic acid molecules remain on the air filter/collectionmedium, or remain in the collection medium after filtering,respectively; isolating said nucleic acid molecules from the airfilter/from the collecting medium or separating the nucleic acidmolecules/cells deposited on the filter/collection medium and subsequentanalysis of the nucleic acid molecules by means of a PCR reaction.

Air Sampling

Air sampling is used to deposit as much airborne nucleic acid aspossible onto a filter surface or in/onto a matrix, for example, asderived from the biogenetic material of a person. Where appropriate, theair volume may be of a significantly high volume. Different air samplingsystems are presently contemplated. In particular, membrane filtrationmay be used. Preferred is an air sampling system employing a suctionpower of at least 1 m³/h, more preferably of at least 3 m³/h. Forexample, the MD 8 isokinetic sampling system from the manufacturerSartorius (see FIG. 2) can be used, wherein the air may be filteredthrough various filter matrices via an intake capacity of 6 m³/h.

The duration of air sampling is dependent on the concentration of theairborne nucleic acids or on the biogenic materials containing them.Preferably, at least 5 m³, at least 10 m³, at least 15 m³, at least 20m³, or at least 25 m³ of air are filtered through the air samplingsystem.

Sampling can be carried out directly using a mobile stand, e.g., at a 1m height positioned centrally in a room. To detect already sedimentedbiogenetic material, it is possible to disrupt any deposited sedimentusing ventilation techniques prior to subjecting this material tofiltration. Alternatively, other known air sampling systems for samplingare contemplated: impaction (exchange of media against filters in emptypetri dishes), impaction on other adsorptive surfaces, impinger methods,electrostatic air collectors, air scrubbers, mobile RLT systems withcollective media such as air filters, etc.

Air Filter/Collecting Media

For the collection medium/air filter, both commercially availablegelatin filters and nitrocellulose matrices are suitable. For gelatinfilters, a pore size of 2 microns to 5 microns, more preferably about 3microns, is preferred. For nitrocellulose filters, a pore size of 5microns to 8 microns is preferred. Other matrices are conceivable,provided they can be adapted to the respective sampling system.

Isolation of Airborne Nucleic Acid Molecules

The isolation of the nucleic acid molecules from the air filters forfurther analysis can be carried out using means known in the art.

For the above-described embodiment relating to forensic analysis, dandere.g. as derived from a crime scene, is deposited on a nitrocellulosefilter via filtration (see FIG. 1, column 1).

The collected dander is then separated from the filter/substratesurface, e.g. using a microscope, and then identified, with eachindividual sample being subjected to further processing.

A second separation technique involves fragmenting the filter/collectionmedium. For this purpose, a device can be used with which thefilter/collection medium can be decomposed into smaller fragments in acontact-free manner using a laser or a mechanical device. Thelaser/mechanical device can be fixed to the filter/collection medium asthe filter/collection medium is, e.g. being moved on an XY cross table.The movement of the XY cross table is achieved via two motor actuators,which can be controlled in using software, e.g. installed on a computer.The complete unit is housed in a sterile environment (e.g. a plexiglasscube with a sterile air supply). Using the described system, the danderfragments obtained from the filter/collection medium are available forfurther analysis.

Following the separation and processing of the collected dander, it ispossible to obtain a complete nucleic acid profile using STR-profilebased multiplex PCR. To this end, the cell membrane should be firstsolubilized by cell lysis. This may occur in 800 μl of a freshlyprepared solution of 0.5 mg/ml lysozyme in TE buffer, pH 8.0. Afteraddition of 80 μl of 10% SDS, the lysate is mixed well and incubated for1-2 min in a water bath at 65° C. This procedure should yield a clear,viscous solution. Subsequently, 88 μl of an 1 M sodium acetate, pH 5.2is added and mixed. The resulting cell lysate is then freed fromproteins using proteinase K and subsequently purified. In the last step,the DNA is concentrated. This may be achieved via ethanol/isopropanolprecipitation. To that end, the solution is mixed with 0.1 volume of an3 M sodium acetate solution (final concentration 0.3 M) and 2 volumes ofcold isopropanol. The precipitated DNA is then sedimented in a table-topcentrifuge and the supernatant discarded. The DNA pellet is washed 1-2times with 75% ethanol and dried. Subsequently, the DNA is taken up indistilled water or sterile buffer. All DNA enrichment/purificationmethods are suitable, including salt precipitation, phenol/chloroformextraction, columns and batch methods.

In another embodiment, nitrocellulose filters are used for the directdetection of free airborne nucleic acid molecules. The filters areeasily extractable using sterile ultrapure water, thus offering thepossibility of direct PCR analysis of the airborne nucleic acids (seeFIG. 1, column 2).

In a further embodiment, cellular bound DNA is also integrated into theanalysis. Here, the entire filter is extracted with a small volume ofwater. A first precipitation is then carried out usingethanol/isopropanol, whereby all free nucleic acids and cells arepelleted via high-speed centrifugation. The pellet is then dissolved,the cells are re-suspended, respectively, in 200 μl of ultrapure water,and the total volume is then used in the further purification steps.Similar to what was described for FIG. 1, column 1, a cell lysis,protein hydrolysis, and a concentration/precipitation is performed (seeFIG. 1, column 3).

In another embodiment, a gelatin filter having a pore size of 3 micronsis used as an air filter. In this embodiment, following filtration, theentire matrix is dissolved in ultrapure water at 43° C., and the gelatinis subsequently removed before PCR, e.g. by using a NucleoSpin Foodextraction kit (Macherey-Nagel). Similar kits from other manufacturersare also suitable. The processing system used in this embodimentprovides, in addition to the removal of all protein contaminants, theadvantage that in addition to the enrichment of pure nucleic acidmolecules, the release and purification of cellular nucleic acid can bealso achieved (see FIG. 1, column 4).

Obtaining a Complete STR Profile from the Isolated Nucleic Acids

An STR (“short tandem repeat”) profile can then be generated for theisolated nucleic acids. For this purpose, in one embodiment, anSTR-multiplex PCR is performed and the resulting amplification productis analyzed using gel electrophoresis and subsequent computer evaluation(see Example 2).

Alternative: Reprocessing of the Analyte Using MALDI-TOF-MS

The analysis of the STR amplification product may also be achieved usingMALDI-TOF mass spectrometry (“matrix assisted laserdesorption/ionization—time off light”).

For this purpose, as already described, amplicons obtained by PCRprocessing (STR regions) are mixed with a concentrated solution of thematrix. Here, the matrix molecules should exhibit an absorption maximumat the wavelength of the irradiated light. For the predominantly usedUV-laser, 3-hydroxypicolinic acid is especially suitable for the DNAanalysis step. After evaporation of the solvent, a semi-crystallinelayer results, whereby the amplicon molecules are ideally completelyseparated from each other via matrix molecules. In the vacuum of themass spectrometer, the amplicon is charged to 10 to 20 kV, and thematrix is then evaporated using laser pulses. An exact determination ofan analyte's molecular mass is possible by accurately measuring the timeit takes for an ion (amplicon) to fly through the field-free, defineddrift region existing between the acceleration electrode and thedetector.

As described below, in obtaining an STR-profile, a profile-to-personassignment is ultimately achieved by means of length polymorphisms atdifferent genetic loci (STR regions) by matching to a database and/or toa control sample.

Alternative: Processing of the Analyte Using in Situ PCR

Additionally, processing of the biogenetic material is possible on thefilter itself. Example 2 relates to a method of generating an STRprofile from dander applied directly on a filter. Here, the geneticmaterial situated on the filter is fixed using paraffin. In subsequentprocessing steps, especially during PCR, it is important to ensure thatthe substances used are protected from evaporation.

EXAMPLES Example 1 Detection of Airborne Nucleic Acid Molecules Using anAir Sampling System

Air sampling systems fitted with air filters were tested to determinewhether they can detect airborne nucleic acid molecules. Here, it isuseful to consider both filter matrix alternatives described above(nitrocellulose and gelatin), especially since the gelatin filters havetemperature- and moisture-related application limitations.

The extraction ability of the filter matrices was determined using“spotting experiments” wherein both filter matrices were loaded withtemplate DNA of a known concentration, followed by extraction of thefilter, and subsequent PCR. The sensitivity criteria of the testedfilter matrices can also be determined by these experiments.

To test the suitability of the filter matrices for the analysis ofairborne nucleic acid molecules, DNA aerosols were generated using aPary-nebulizer, whose particle sizes are previously defined by particlemonitoring. The aerosols were supplied by a ventilation system (airvolume flow rate 300 m³/h) of the isokinetic sampling air using membranefiltration using a Sartorius MD 8 system. The filters were then treatedaccording to the following processing scheme, and then subjected to PCR.

The PCR samples were subjected to agarose gel electrophoresis foranalysis, labeled using SYBR® Gold, and then photographed on atransilluminator. A standard size marker (250-10,000 bp) from Invitrogenwas used.

Example 2 Obtaining a Complete STR Profile from Dander

Isolation of DNA

In order to obtain a sufficient amount of dander, air from a crime sceneenvironment was first collected for a 3 hour period and filtered througha nitrocellulose filter at 6 m³/h using a Sartorius MD 8 air samplingsystem. During the filtering, usually air from that specific room roomis collected, but it is conceivable that the sampling of air from otherindoor spaces may occur, e.g., air derived from nearby vehicles. Eachobtained dander sampling was individually transferred from the filterwith the aid of a light microscope and a micromanipulator under sterileconditions in a separate reaction vessel. Following the addition of 55μl Chelex solution, 5% (Bio-Rad Laboratories, Hercules, Calif., USA) and3 μl of proteinase K (10 IU/ml) (QIAGEN GmbH, Hilden, Germany), thesamples were incubated in a thermal mixer at 37° C. and 450 rpmovernight (ca. 14 h). The samples were then boiled for 8 minutes at 93°C. and centrifuged at 13,000 rpm for 15 minutes. The Chelex-granule-freesupernatant contained the DNA to be isolated.

STR Multiplex PCR

To select for successful extraction, 7 μl of each extract was used in amultiplex PCR with the cost-effective and highly sensitive Kit Q8(in-house Short Tandem Repeat-PCR kit, University of Ulm, availablewithout prescription) for 33 cycles using the following profile:

Hot start: 11 min at 95° C.

Denaturation: 1 min at 93° C.; 33 cycles

Annealing: 1 min at 59° C.

Elongation: 30 s at 72° C.

Final Elongation: 40 min at 72° C.

The Kit Q8 is a miniSTR-Multiplex-PCR kit containing shortened ampliconsfor the eight different German database systems D3S1358, FGA, TH01, VWA,SE33, D8S1179, D18551 and D21511 as well as the gender markeramelogenin. The following approach for a total volume of 10.9 μl each isused per reaction:

1.6 mM MgCl₂ (Applied Biosystems)

0.3 μl BSA (Boehringer Mannheim GmbH)

1.25 μl PCR buffer II (Applied Biosystems)

each 0.1 mM dNTPs

0.75 μl primer mix

2 U Ampli Taq Gold (Applied Biosystems)

1-7 μl DNA template

0-6 μl double-distilled water

The subsequent purification of the PCR product was performed using the“QIAquick® PCR Purification Kit” (Qiagen, Hilden, Germany) according tothe manufacturer's protocol. This system almost completely removes allenzymes, salts, oligomers, excess primers, nucleotides and various otherforeign materials from the sample, and concentrates double-stranded DNAwith a length of between 70 bp and 4 kb to a final volume of 9 μl.

For the electrophoresis, using an ABI Prism 3130 Genetic Analyzer(Applied Biosystems), each sample well was loaded with 10 μl Hidi™formamide (Applied Biosystems), 0.5 μl standard GeneScan™ 500 ROX sizestandard (SERAC, Bad Homburg, Germany), and 1 amplicon, and boiled for 4min at 94° C. Electrophoresis was carried out with the polymer POP7(Applied Biosystems) with an injection time of 16 s, an injectionvoltage of 1.2 kV and a duration of 800 s. For the subsequent analysis,e.g. the software GeneMapperID v3.2 (Applied Biosystems) can be used.

Samples where not a single expected allele was detected are counted asempty. Samples containing more than two of the expected alleles, bycontrast, are classified as useful for further investigation. For thosesamples where one or two of the expected alleles was detected, 7 μlextract is removed and subjected to a second round with the Q8-Kit for afurther 33 cycles. If no correct allele was detected during the secondround, the sample was also considered to be empty, but, on the otherhand, where at least one correct allele was detected, this sample wassubjected to subsequent processing by RT-PCR.

Quantification by Means of RT-PCR

For quantification using real-time PCR, the commercially available kitPlexor® HY (Promega) was applied on a 7500 Real Time PCR System (AppliedBiosystems).

The Plexor® HY system is based on the interaction between two modifiednucleotides. One of the PCR primers contains a nucleotide having afluorescent iso-dC label. If the PCR is successful, the modifiedDabcyl-iso-dGTP also contained in the PCR reaction mix, anneals to theabove-mentioned primer and when inserted into the complementary DNAstrand, leads to a decrease in fluorescence. By using different labelsand primer sets, quantification of human DNA and human male DNA, and anassessment of possible inhibitors by an internal PCR control (IPC), issimultaneously possible. The target sequence of the autosomal primerlabeled with the fluoresceine marker is a 99 bp long multi-copy sequenceon chromosome 17 in the human RNU2 locus, which encodes an snRNA whichis involved in the pre-mRNA processing. The Y-chromosomal primer is CALFluor® Orange 560-labeled and amplifies a 133-bp sequence of thetestis-specific protein, which is part of the YSPY locus. CAL Fluor® 610RED is a fluorescence marker for the IPC, IC5 is also included as afourth label in all wells as a control reference. The 6.4 pg to 100 ngcontrol standard enables a sensitivity sufficient to validate thequantification in the above-mentioned range. The implementation followsthe requirements specified in the technical manual for an increasedetectable in a sample extract volume from 2 to 3 μl. Accordingly, foreach reaction, 10 μl of master mix, 1 μl primer/IPC Mix, 6 μldouble-distilled water and 3 μl template DNA is used. Each sample wasquantified in duplicate using two control standards and two emptycontrols per run. The PCR program consists of an initial denaturation at95° C. for 2 min with subsequent 38 cycles of a 5 sec denaturation at95° C. and a 35 s annealing and elongation at 60° C. Following the PCTprotocols, the generated melting curves allows a control of thespecificity of resulting products.

The invention claimed is:
 1. A method for analyzing airborne nucleicacid molecules in airborne human cellular material, the methodcomprising: filtering and/or collecting air in an air sampling systemthat comprises an air filter and/or a collection medium, such that thenucleic acid molecules in the filtered/collected cellular materialremain on the air filter and/or in the collection medium; isolating thenucleic acid molecules in the filtered/collected cellular material fromthe air filter and/or from the collecting medium; analyzing the isolatednucleic acid molecules; and obtaining a human DNA profile.
 2. The methodaccording to claim 1, wherein the air filter is a nitrocellulosemembrane filter.
 3. The method of claim 2, wherein the nitrocellulosemembrane filter includes a pore size of between 5 μm to 8 μm.
 4. Themethod according to claim 1, wherein the air filter is a gelatin filter.5. The method according to claim 4, wherein the gelatin filter includesa pore size of between 2 μm to 5 μm.
 6. The method according to claim 1,wherein the collection medium is a buffer for collecting the cellularmaterial by impaction.
 7. The method according to claim 1, wherein thecollection medium is a surface on which the cellular material isdeposited electrostatically.
 8. The method according to claim 4, whereinthe filter/collection medium is decomposed into individual fragments ona contact-free basis by means of a laser or mechanical device situatedin a sterile housing unit, whereby the individual fragments areprocessed manually or automatically.
 9. The method according to claim 1,wherein the air sampling system is a device for isolating non-gaseousconstituents from the air.
 10. The method according to claim 9, whereinthe air sampling system is a Sartorius MD 8 system, an impaction systemor an electrostatic system.
 11. The method according to claim 1, whereinthe filtering and/or collecting air is performed in a laboratory room, aproduction room, a storage room, a lounge, a meeting room or a crimescene.
 12. The method according to claim 1, wherein the filtering and/orcollecting air in the air sampling system occurs at a suction power ofat least 1 m³/h.
 13. The method according to claim 12, wherein the airsampling system is the Sartorius MD 8 system and the suction power is 6m³/h.
 14. The method according to claim 1, wherein at least 5 m³ of airis filtered and/or collected through the air sampling system.
 15. Themethod according to claim 1, wherein the filtering and/or collecting airis carried out using impaction, impinging methods, electrostatic aircollectors, air scrubbers, or mobile RLT systems with collective media.